Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
  • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/36—Gynecology or obstetrics
  • G01N2800/362—Menopause

Definitions

• This disclosure relates generally to neurological and endocrinological disease, and more particularly, to the identification of peripherin-specific antoantibodies as a marker for such neurological and endocrinological disease.
• Neural-restricted autoantibodies are emerging as serum biomarkers of acquired neurological disorders, both idiopathic and paraneoplastic.
• Autoantigens identified to date are expressed in neurons, glia or skeletal muscle.
• Plasma membrane autoantigens are potential targets of pathogenic IgG and are exemplified by channel proteins. These include neuronal voltage-gated potassium channels and NMDA and AMPA receptors in encephalopathies, astrocytic water channels in neuromyelitis optica, muscle acetylcholine receptors [AChR] in myasthenia gravis and neuronal AChR in autoimmune autonomic ganglionopathies.
• Antibodies recognizing intracellular autoantigens are not pathogenic to intact cells.
• IgGs reactive with neuronal and glial cytoplasmic and nuclear proteins such as glutamic acid decarboxylase-65 (GAD65) in stiff-man syndrome, cerebellitis and encephalomyelopathy, and anti-neuronal and glial nuclear autoantibodies in multifocal paraneoplastic disorders.
• GID65 glutamic acid decarboxylase-65
• Peripherin-specific autoantibodies have been identified as a serum biomarker in individuals with autoimmune neurological disorders (particularly targeting the peripheral autonomic and somatic nervous system, spinal cord and optic nerves) often in the context of endocrinological disease, and sometimes with cancer.
• the detection of peripherin- specific autoantibodies can be used to support the diagnosis of an individual with autoimmune neurological and endocrinological disease.
• a method of detecting the presence or absence of a peripherin-specific autoantibody in a biological sample from an individual is provided.
• Such a method generally includes the steps of contacting the biological sample with a peripherin polypeptide or fragment thereof; and detecting the presence or absence of binding of the peripherin polypeptide or fragment thereof to the peripherin-specific autoantibody in the biological sample.
• the presence of the peripherin-specific autoantibody in the biological sample is associated with dysautonomia or small fiber neuropathy
• the peripherin polypeptide or fragment thereof is in a cell lysate; in other embodiments, the peripherin polypeptide is in a solid tissue selected from the group consisting of brain (e.g., hindbrain), kidney, stomach or other tissues containing peripheral nerve elements.
• dysautonomia include gastrointestinal (GI) dysmotility, abnormal sudomotor function, abnormal cardiovagal function, or abnormal adrenergic function.
• endocrinopathy include diabetes, thyroid disorders, or premature menopause.
• neurological manifestations include transverse myelitis, non-specified myelopathy, optic neuropathy, encephalitis, lumbosacral plexopathy, sensorimotor neuropathy, cerebellar ataxia or myasthenia gravis.
• the biological sample is selected from the group consisting of serum, plasma, cerebrospinal fluid, and blood.
• an article of manufacture in another aspect, generally includes a peripherin polypeptide or fragment thereof and instructions for using the peripherin polypeptide to detect an anti-peripherin autoantibody in an individual.
• the article of manufacture is used to diagnose the presence or absence of a peripherin-associated autoimmune disease in the individual.
• an article of manufacture further includes a monoclonal antibody having specific binding affinity for a peripherin polypeptide or fragment thereof.
• a method of treating an individual having a peripherin- associated autoimmune disease typically includes withdrawing a body fluid from the individual, wherein the body fluid contains one or more autoantibodies that bind to peripherin; removing a substantial portion of the autoantibodies from the body fluid; and returning the body fluid to the subject.
• a method of treating an individual having a peripherin- associated autoimmune disease typically includes administering a peripherin polypeptide to the individual.
• administration is by a method selected from the group consisting of orally, intravenously, and parenterally.
• Figure 1 is the nucleotide (SEQ ID NO: l) and amino acid (SEQ ID NO:2) sequence of human peripherin.
• Figure 2 is the nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequence of mouse peripherin.
• Figure 3 are photographs demonstrating that a novel IgG autoantibody binds to neural elements in sections of mouse stomach and kidney and to discrete fiber tracts in mid-hind brain. Bound IgG was visualized using fluorescein-conjugated anti-human IgG. The characteristic staining pattern of this autoantibody is prominent in myenteric ganglia, nerve fibers and nerve trunks in the enteric nervous system (upper panel), sympathetic nerve trunks and fibers adjacent to arterioles in the gastric submucosa and kidney (center panel) and discrete nerve bundles in the mid-hind brain (lower panel).
• G ganglion
• NT nerve trunk
• NF nerve fibers
• SN sympathetic nerve.
• Figure 4 are photographs demonstrating that the antigen associated with the novel autoantibodies described herein is restricted to neurons.
• Differentiated cell lines used as substrates pheochromocytoma (PC 12; left panels), astrocytes (CG4; middle panels) and skeletal muscle (L6; right panels).
• Informative IgG probes included:
• Neurofilament neurovascular endothelial
• GFAP glial
• striational skeletal muscle sarcomere
• Figure 5 are results demonstrating that the novel IgG autoantibody binds to a protein having a Mr of 55-60 kDa that is confined to the neuronal cytoskeleton.
• A Using PC 12 lysate as a source of antigen, proteins were separated, immunob lotted, and probed with individual patient or normal human sera. A common band ( ⁇ 55 kDa by reference to molecular weight standards) was revealed by IgG in individuals' sera (lanes 1-4), but not by IgG in control human serum (lanes 5-6). To verify specificity, individual IgG was affinity-purified on the putative antigenic band and a control band.
• Eluates from the putative antigenic band (B) and control band (C) were re-applied to the composite mouse tissue substrate slide, and compared to the original immunostaining pattern of whole individual serum IgG on enteric nerve fibers (D). An identical staining pattern was observed in eluates from the putative antigenic band.
• PC 12 lysates were fractionated by differential detergent extraction. Proteins in each fraction were resolved electrophoretically, transb lotted, and probed with individual sera (E). IgG bound exclusively to a protein in the cytoskeletal fraction.
• Figure 6 are results that identify peripherin as the antigen.
• Duplicate preparations of the cytoskeletal fraction of PC 12 cells were separated by sequential gel electrophoresis and isoelectric focusing. One gel was silver stained (A), and the replica was transblotted and probed with individual serum (B). Unique peptides yielded by in-gel digestion from the silver stained gel of the common spots (numbered 1-4) identified the antigen to be peripherin.
• FIGS. 7 are photographs that demonstrate that individual IgG colocalizes with peripherin immunoreactivity in brain and endocrine organs.
• Tissues (brain [A-C], thyroid [D-F], pancreas [G-I], ovary [J-L] and liver [M-O]) were harvested from a 6-8 week old female mouse, cryosections (8 ⁇ ) were cut and stained with rabbit anti- peripherin-IgG (left columns) and individual patient IgG (center columns).
• a specific IgG autoantibody marker has been identified in serum of individuals presenting with dysautonomia (e.g., gastrointestinal (GI) dysmotility, abnormal sudomotor function, abnormal cardiovagal function or abnormal adrenergic function), endocrinopathy (e.g., diabetes, thyroid disorders, or premature menopause) and other neurological disorders (e.g., particularly myelopathies, optic and other cranial neuropathies, cerebellar ataxia and sensory-predominant neuropathies but also lumbosacral plexopathy, encephalitis, or myasthenia gravis).
• dysautonomia e.g., gastrointestinal (GI) dysmotility, abnormal sudomotor function, abnormal cardiovagal function or abnormal adrenergic function
• endocrinopathy e.g., diabetes, thyroid disorders, or premature menopause
• other neurological disorders e.g., particularly myelopathies, optic and other cranial neuropathies, cerebellar ataxia and sensory-pre
• peripherin a type III neuronal intermediate filament protein that forms networks, either alone or complexed with other neurofilament proteins. It is attributed a role in neuron development and repair, and is distributed widely in the peripheral nervous system. In the central nervous system, it is restricted to regions that project to the periphery.
• this disclosure provides for methods of detecting peripherin-specific autoantibodies in an individual that presents with dysautonomia, myelopathy, vision impairment, endocrinopathy, sensory-predominant neuropathies or other neurological disorders. In a diabetes environment, the presence of peripherin-specific autoantibodies may be used to evaluate the individual's risk for developing neurological complications (e.g., small fiber neuropathy).
• neurological complications e.g., small fiber neuropathy
• peripherin-specific autoantibodies may be used to evaluate the individual's potential to benefit from immunosuppressant therapies.
• Peripherin Polypeptides and Anti-Peripherin Antibodies may be used to evaluate the individual's potential to benefit from immunosuppressant therapies.
• Peripherin polypeptides can be used in the methods described herein (e.g., detecting a peripherin-specific autoantibody).
• Examples of peripherin polypeptide sequences (and the nucleic acids encoding such polypeptides) can be found in GenBank Accession Nos. BC046291; NM_001001235; NM_006262; NM_012633; BC100656; NM 001087060; and NM l 31054. Additional peripherin sequences can be found, for example, in public databases.
• a representative human peripherin sequence is shown in Figure 1 (SEQ ID NOs: 1 and 2; DNA and protein, respectively), and a representative mouse peripherin sequence is shown in Figure 2 (SEQ ID NOs: 3 and 4; DNA and protein, respectively).
• Peripherin polypeptides like other intermediate filaments, are insoluble.
• peripherin polypeptides are well-studied, and there are many reports in the literature that describe methods of working with peripherin. See, for example, Portier et al. (1983-4, Dev. NeuroscL, 6(6):335-44); Landon et al. (2000, Biol. Cell, 92(6):397- 407); McLean et al. (2008, J. Neurochem., 104(6): 1663-73); Puertas et al. (2007, J. Immun., 178(10):6533-39); and Aletta et al. (1989, J. Biol. Chem., 264(8):4619-27). In addition, those skilled in the art are aware of methods that can be used to increase the solubility of a polypeptide. See, for example, Trimpin & Brizzard, 2009, BioTechniques, 46(6):409-19.
• Peripherin polypeptides may be obtained from human, mouse or other
• polypeptides can be purified.
• a "purified" polypeptide refers to a polypeptide that constitutes the major component in a mixture of components, e.g., 30% or more, 40% or more, 50%> or more, 60%> or more, 70%> or more, 80%> or more, 90%> or more, 95% or more, or 99% or more by weight.
• Polypeptides may be purified by methods including affinity chromatography or immunosorbant affinity column. Such methods can be modified by those skilled in the art to increase the solubility of the polypeptide, and purified polypeptides can be examined for their immunogenicity using routine methods.
• peripherin polypeptide sequence see, for example, SEQ ID NOs: 2 and 4
• any polypeptide fragment can be generated by known means (e.g., proteolytic cleavage of a polypeptide or chemical synthesis). It would be understood by those skilled in the art that a fragment of peripherin may have a different solubility than that of the full-length peripherin polypeptide.
• Fragments of a peripherin polypeptide can contain one or more epitopic sites. Epitopic sites within peripherin polypeptides that are pertinent to T-cell activation and suppression (e.g., MHC-I and MHC-II binding epitopes) can be determined by direct investigation, or by using computer algorithms.
• the epitopic site be antigenically distinct from other closely related antigens (e.g., other members of a family of polypeptides).
• Peripherin polypeptides or fragments thereof may be used with or without modification for the detection of peripherin-specific antibodies such as peripherin- specific autoantibodies.
• Polypeptides can be labeled by either covalently or non- covalently combining the polypeptide with a second substance that provides for detectable signal.
• labels and conjugation techniques are known in the art and are reported extensively in both the scientific and patent literature. Some of the labels include radioisotopes, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles, and the like.
• Peripherin polypeptides or fragments thereof can be used in various ways.
• peripherin polypeptides can be used in an immunoassay to detect peripherin-specific autoantibodies in a biological sample.
• Peripherin polypeptides used in an immunoassay can be in a cell lysate (e.g., a whole cell lysate or a cell fraction), or purified peripherin polypeptides or fragments thereof can be used provided at least one antigenic site recognized by peripherin-specific antibodies (e.g., peripherin-specific autoantibodies) remains available for binding.
• a cell lysate e.g., a whole cell lysate or a cell fraction
• purified peripherin polypeptides or fragments thereof can be used provided at least one antigenic site recognized by peripherin-specific antibodies (e.g., peripherin-specific autoantibodies) remains available for binding.
• either or both immunoassays and immunocytochemical staining techniques may be used.
• Enzyme- linked immunosorbent assays ELISA
• Western blot Western blot
• radioimmunoassays are methods used in the art, and can be used as described herein to detect the presence of peripherin-specific autoantibodies in a biological sample.
• a “biological sample,” as used herein, is generally a sample from an individual.
• biological samples include blood, serum, plasma, or cerebrospinal fluid.
• solid tissues for example, spinal cord or brain biopsies may be used.
• kits containing one or more peripherin polypeptides or fragments thereof.
• Peripherin polypeptides or fragments thereof that are included in an article of manufacture as described herein can be provided within a cell, in a solution in which they are soluble, or the peripherin polypeptides or fragments thereof can be provided in a lyophilized form.
• an article of manufacture as described herein also can include one or more compounds for increasing the solubility of a polypeptide (e.g., a solubilizing agent).
• the kit may further include a second substance that, for example, provides for a detectable signal.
• a kit can include directions for using the peripherin polypeptides and/or directions for practicing a method described herein (i.e., detecting peripherin-specific autoantibodies in a biological sample).
• the present disclosure also provides for methods of detecting peripherin polypeptides. Detection of a polypeptide is typically performed using an antibody, referred to herein as an anti-peripherin antibody to distinguish such animal- or recombinantly-generated antibodies from peripherin-specific autoantibodies produced by an individual's immune system.
• an antibody including a monoclonal antibody, with specific binding affinity for peripherin polypeptides or antigenic fragments thereof.
• Peripherin polypeptides as described herein can be used to produce monoclonal or polyclonal anti-peripherin antibodies having specific binding affinity for the peripherin polypeptide. Such antibodies can be produced using techniques known to those of ordinary skill in this art.
• anti-peripherin antibodies having "specific binding affinity" for peripherin polypeptides or fragments thereof are defined as those antibodies that preferentially bind peripherin polypeptides or fragments thereof, but that do not bind or have very little affinity for non-peripherin polypeptides.
• anti-peripherin antibody can be whole antibodies of any class (e.g., IgG, IgA, IgM), portions or fragments of whole antibodies (e.g., Fab or (Fab) 2 fragments) having the desired specific binding affinity, an engineered single chain Fv molecule, or a chimeric molecule, e.g., an antibody that contains the binding specificity of one antibody (e.g., of murine origin) and the remaining portions of another antibody (e.g., of human origin).
• IgG IgG, IgA, IgM
• portions or fragments of whole antibodies e.g., Fab or (Fab) 2 fragments
• an engineered single chain Fv molecule e.g., an antibody that contains the binding specificity of one antibody (e.g., of murine origin) and the remaining portions of another antibody (e.g., of human origin).
• Anti-peripherin antibodies may be used with or without modification for the detection of peripherin polypeptides.
• Anti-peripherin antibodies can be labeled either directly or indirectly, and a wide variety of labels, including radioisotopes, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers and magnetic particles, and conjugation techniques are known and are reported extensively in both the scientific and patent literature.
• an anti-peripherin antibody having specific binding affinity for peripherin polypeptides conjugated to an imaging agent. Suitable imaging agents include, but are not limited to, radioisotopes, such as 32 P,
• Anti-peripherin antibodies as described herein can be used in various combinations
• immunological techniques for detecting peripherin polypeptides The use of antibodies in protein binding assays is well established. Depending on the nature of the sample, immunoassays (e.g., radioimmunoassays) and/or immunohistochemical /
• Liquid phase immunoassays e.g., competitive inhibition radioimmunoassays
• solid phase immunoassays e.g., antigen-capture or Western blot analysis
• ELISA enzyme-linked immunosorbent assays
• kits containing anti-peripherin antibodies having binding affinity for peripherin polypeptides or fragments thereof may also include peripherin polypeptides or fragments thereof to be used as binding controls or to generate a standardized quantitative curve.
• the kit may further include a second substance that provides for detectable label.
• a kit typically includes directions for using an anti-peripherin antibody (e.g., for detecting or purifying peripherin polypeptides).
• nucleic acid refers to RNA or DNA.
• isolated refers to (i) a nucleic acid sequence encoding part or all of peripherin polypeptide, but free of coding sequences that normally flank one or both sides of the nucleic acid sequences encoding peripherin in the genome; or (ii) a nucleic acid incorporated into a vector or into the genomic DNA of an organism such that the resulting molecule is not identical to any naturally-occurring vector or genomic DNA.
• Peripherin nucleic acids are shown in Figures 1 and 2 (SEQ ID NO:l and 3), as well as in the GenBank Accession Nos. provided herein.
• Peripherin nucleic acids also can include fragments of peripherin nucleic acid sequences.
• fragments refer to nucleic acids or polypeptides corresponding to less than an entire peripherin sequence.
• Nucleic acid fragments may include those fragments of about 10 to 50 nucleotides in length, fragments of about 20 to 100 nucleotides in length, or fragments that are 100 to several hundred nucleotides in length. Such fragments may, for example, encode a peripherin polypeptide fragment, or have utility as hybridization probes or amplification primers.
• nucleic acid fragment Given a peripherin nucleotide sequence (see, for example, SEQ ID NOs: 1 and 3), virtually any nucleic acid fragment can be generated by known means (e.g., restriction enzyme digestion, the polymerase chain reaction) and, if so desired, expressed to produce the corresponding polypeptide fragment.
• Various restriction enzyme sites within a peripherin nucleic acid sequence define positions that, in various combinations, can be used to generate useful nucleic acid fragments.
• a peripherin nucleic acid or nucleic acid fragment may have a sequence that deviates from a wild type peripherin sequence (e.g., SEQ ID NOs: 1 and 3), sometimes referred to as a variant sequence.
• a variant nucleic acid sequence can have at least 80% sequence identity to a wild type peripherin sequence (e.g., SEQ ID NOs: 1 and 3).
• the variant nucleic acid sequence can have at least 85% sequence identity, 90% sequence identity, 95% sequence identity, or at least 99% sequence identity to a wild type peripherin sequence (e.g., SEQ ID NOs: 1 and 3).
• Variant nucleic acid sequences can be designed that encode variant peripherin polypeptides or fragments thereof that, for example, exhibit increased solubility compared to the wild type polypeptides or fragments thereof.
• Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid or polypeptide sequences, dividing the number of matched positions by the total number of aligned nucleotides or amino acids, respectively, and multiplying by 100.
• a matched position refers to a position in which identical nucleotides or amino acids occur at the same position in aligned sequences.
• the total number of aligned nucleotides or amino acids refers to the minimum number of peripherin nucleotides or amino acids that are necessary to align the second sequence, and does not include alignment (e.g., forced alignment) with non-peripherin sequences, such as those fused to peripherin.
• the total number of aligned nucleotides or amino acids may correspond to the entire peripherin sequence or may correspond to fragments of the full-length peripherin sequence as defined herein.
• Sequences can be aligned using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389-3402) as incorporated into BLAST (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web.
• BLAST searches or alignments can be performed to determine percent sequence identity between a peripherin nucleic acid molecule and any other sequence or portion thereof using the Altschul et al. algorithm.
• BLASTN is the program used to align and compare the identity between nucleic acid sequences
• BLASTP is the program used to align and compare the identity between amino acid sequences.
• a nucleic acid encoding a peripherin polypeptide may be obtained from, for example, a cDNA library made from a human or rat cell line, or can be obtained by other means, including, but not limited to, the polymerase chain reaction (PCR).
• PCR refers to a procedure or technique in which target nucleic acids are amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
• Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
• Peripherin nucleic acids can be detected by methods such as Southern or Northern blot analysis (i.e., hybridization), PCR, or in situ hybridization analysis. Hybridization between nucleic acid molecules is discussed in detail in Sambrook et al. (1989,
• oligonucleotide probes less than about 100 nucleotides For oligonucleotide probes less than about 100 nucleotides, Sambrook et al. discloses suitable Southern blot conditions in Sections 11.45-11.46. The Tm between a sequence that is less than 100 nucleotides in length and a second sequence can be calculated using the formula provided in Section 11.46. Sambrook et al. additionally discloses prehybridization and hybridization conditions for a Southern blot that uses oligonucleotide probes greater than about 100 nucleotides (see Sections 9.47-9.52). Hybridizations with an oligonucleotide greater than 100 nucleotides generally are performed 15-25°C below the Tm.
• the Tm between a sequence greater than 100 nucleotides in length and a second sequence can be calculated using the formula provided in Sections 9.50-9.51 of Sambrook et al. Additionally, Sambrook et al. recommends the conditions indicated in Section 9.54 for washing a Southern blot that has been probed with an oligonucleotide greater than about 100 nucleotides. The conditions under which membranes containing nucleic acids are
• prehybridized and hybridized as well as the conditions under which membranes containing nucleic acids are washed to remove excess and non-specifically bound probe can play a significant role in the stringency of the hybridization.
• Such hybridizations can be performed, where appropriate, under moderate or high stringency conditions. Such conditions are described, for example, in Sambrook et al. section 1 1.45-1 1.46.
• washing conditions can be made more stringent by decreasing the salt concentration in the wash solutions and/or by increasing the temperature at which the washes are performed.
• interpreting the amount of hybridization can be affected, for example, by the specific activity of the labeled oligonucleotide probe, by the number of probe-binding sites on the template nucleic acid to which the probe has hybridized, and by the amount of exposure of an autoradiograph or other detection medium.
• hybridization and washing conditions can be used to examine hybridization of a probe nucleic acid molecule to immobilized target nucleic acids, it is more important to examine hybridization of a probe to target nucleic acids under identical hybridization, washing, and exposure conditions.
• the target nucleic acids are on the same membrane.
• a nucleic acid molecule is deemed to hybridize to a first target nucleic acid but not to a second target nucleic acid if hybridization to the first nucleic acid is at least 5- fold (e.g., at least 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater than hybridization to the second nucleic acid.
• the amount of hybridization can be quantitated directly on a membrane or from an autoradiograph using, for example, a Phosphorlmager or a Densitometer (Molecular Dynamics, Sunnyvale, CA).
• vectors containing a peripherin nucleic acid see, for example, SEQ ID NOs: 1 and 3) or the complements thereof, peripherin nucleic acid fragments or the complements thereof, and those nucleic acids having at least 80% sequence identity to a peripherin nucleic acid or fragments generated therefrom (or the complements thereof).
• Cloning vectors are commercially available and used routinely by those of ordinary skill.
• Vectors may additionally comprise elements necessary for expression operably linked to a peripherin nucleic acid sequence.
• “Elements necessary for expression” include promoter sequences, and additionally may include regulatory elements, such as enhancer sequences, response elements or inducible elements that modulate expression of a peripherin nucleic acid sequence.
• operably linked refers to positioning of a promoter and/or other regulatory element(s) in a construct relative to a peripherin nucleic acid sequences in such a way as to direct or regulate expression of the peripherin nucleic acid.
• constructs are commercially available (e.g., expression vectors) and/or produced by recombinant DNA technology methods routine in the art.
• the choice of expression systems depends upon several factors, including, but not limited to, replication efficiency, selectability, inducibility, targeting, the level of expression desired, ease of recovery and the ability of the host to perform post-translational modifications.
• Cloning vectors are available that increase the solubility of the expressed polypeptide. See, for example, U.S. Patent Nos. 7,501,484 and 7,524,648.
• host or "host cell” is meant to include not only prokaryotes, such as E. coli, but also eukaryotes, such as yeast, insect, plant and animal cells.
• Animal cells include, for example, COS cells and HeLa cells.
• a host cell can be transformed or transfected with a DNA molecule (e.g., a vector or construct) using any of the techniques commonly known to those of ordinary skill in this art, such as calcium phosphate or lithium acetate precipitation, electroporation, lipofection and particle bombardment.
• Host cells containing a vector as described herein may be used for purposes such as propagating the vector, producing peripherin nucleic acid (e.g., DNA, RNA, antisense RNA), or expressing a peripherin polypeptide or fragments thereof.
• peripherin nucleic acid e.g., DNA, RNA, antisense RNA
• Methods of producing peripherin polypeptides from peripherin nucleic acids are provided.
• Methods of producing peripherin polypeptides include, but are not limited to, culturing host cells containing a peripherin expression vector under conditions permissive for expression of peripherin, and recovering (e.g., purifying) the peripherin polypeptides.
• Methods of culturing bacteria and recovering expressed polypeptides, including insoluble polypeptides are well known to those of ordinary skill in this art.
• Treatment of such a peripherin-associated autoimmune disease can include, without limitation, apheresis and/or T cell receptor-based immunotherapy.
• This disclosure provides a method of removing peripherin-specific autoantibodies from a body fluid of an individual.
• the method involves withdrawing a body fluid from a subject; removing a substantial portion of peripherin-specific autoantibodies from the fluid; and returning the fluid to the subject.
• Antibodies removed can be of any class, e.g., IgG (such as IgGl, IgG2, IgG3, IgG4), IgM, IgD, IgA, or IgE antibodies.
• a "substantial portion” means removing at least 20% (e.g., at least: 20%; 30%; 40%; 50%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 93%; 95%; 96%; 97%; 98%; 99%; 99.5%; 99.8%; or even 100%) of the peripherin-specific autoantibodies that were present in the body fluid prior to removal.
• the body fluid can be blood plasma or any other body fluid, e.g., lymph or cerebrospinal fluid.
• depleting peripherin-specific autoantibodies from individuals with a peripherin-associated autoimmune disease may result in a reduction or a decrease in one or more of the symptoms.
• Removal of peripherin-specific autoantibodies is generally performed by contacting a body fluid with a peripherin polypeptide or fragment thereof.
• the peripherin polypeptide or fragment thereof can be bound to a solid support.
• Such solid supports can be, without limitation, membranes, fibers, spherical beads, or granules and can be made with a water-insoluble, preferably porous, biocompatible material, e.g., organic polymers such as agarose, dextran, and polyacrylamide, or inorganic porous materials such as porous glass or porous silica gel.
• Such materials are suitable or can be adapted (e.g., derivatized with appropriate chemical groups) for attachment of a peripherin polypeptide.
• the plasma and/or white blood cells can be separated from red blood cells (e.g., erythrocytes) and the red blood cells can be returned to the individual with or without white blood cells.
• red blood cells e.g., erythrocytes
• the blood cells are returned to the individual with artificial rather than their original blood plasma.
• the "replacement fluid" e.g., physiological saline
• the peripherin-specific autoantibodies can be selectively removed from the blood plasma in the course of apheresis and the blood cells can be mixed with the peripherin-specific autoantibody-depleted plasma and then re-infused as a mixture into the individual.
• the system can be a continuous one in which, for example, blood is pumped out of a blood vessel (e.g., an artery or a vein) passed over a solid support derivatized with peripherin polypeptides and pumped directly back into a blood vessel of the subject.
• a blood vessel e.g., an artery or a vein
• a solid support derivatized with peripherin polypeptides and pumped directly back into a blood vessel of the subject.
• blood cells can be separated from plasma prior to passing of the plasma over the solid support.
• T cell receptor therapy In addition to apheresis, methods of T cell receptor therapy are known in the art.
• Monoclonal or polyclonal antibodies having specific binding affinity for the antigen(s) expressed by the peripherin-receptor or other marker on the T cell population responsible for inducing and maintaining the production of peripherin-specific autoantibodies can be used to deplete or suppress one or more pathogenic T cells.
• CDR3 spectratyping of T cell receptors can be used to identify autoimmune disease-associated T cell receptors (Matsumoto et al, supra; and Jambou et al, 2003, J. Clin. Invest., 112:254- 74).
• activation of T cells can be inhibited in an individual by administering a cytokine or an antibody having specific binding affinity for a cytokine.
• a cytokine such as interleukin (IL)-4, IL-10, or IL-13, or an antibody specific for a cytokine such as IL-12 or IFN- ⁇ can be administered to an individual.
• a cytokine such as IL-12 or IFN- ⁇ or an antibody specific for IL-4, IL-10, or IL-13 can be administered to an individual.
• a cytokine such as IL-12 or IFN- ⁇ or an antibody specific for IL-4, IL-10, or IL-13 can be administered to an individual.
• methods of enumerating or isolating peripherin-specific T-cells from an individual This method may be used, for example, to monitor an individual's immune response or for immunotherapy using peripherin-specific cytotoxic T-cells.
• the method comprises contacting a biological sample containing lymphocytes with tetrameric soluble class I or class II major histocompatibility complex (MHC) bearing identical peripherin polypeptides or fragments thereof.
• MHC major histocompatibility complex
• Linker molecules such as avidin and biotin are used to produce the peripherin polypeptide-MHC tetrameric complex, which can subsequently be labeled with an indicator molecule such that those T-cells that recognize the peripherin polypeptide-MHC tetrameric complex are enumerated or isolated (e.g., using FACS analysis). See, for example, Schwartz, 1998, New England J. Med., 339: 1076-8, and references therein.
• therapeutic methods can include administering an effective amount of a pharmaceutical composition (e.g., a peripherin polypeptide or a nucleic acid such as an antisense oligonucleotide or a nucleic acid encoding a peripherin polypeptide) to an individual.
• a pharmaceutical composition e.g., a peripherin polypeptide or a nucleic acid such as an antisense oligonucleotide or a nucleic acid encoding a peripherin polypeptide
• An effective amount is an amount that deviates the individual's peripherin-mediated immune response, thereby modulating the autoimmune disease in the individual.
• modulating an autoimmune disease can refer to reducing the severity of one or more symptoms, eliminating all symptoms, or any level of symptoms therebetween.
• Peripherin polypeptides can be delivered to an individual directly or by administering a vector appropriately expressing a nucleic acid encoding a peripherin polypeptide.
• Vectors for delivering nucleic acids that encode biologically useful proteins to an individual are known in the art and include, for example, adenovirus, adeno- associated virus, retroviruses, lentiviruses, vaccinia virus, herpes viruses, and bovine papilloma virus nucleic acids.
• administering refers to a method of delivering a composition (e.g., a peripherin polypeptide, a nucleic acid encoding a peripherin polypeptide, or an antisense oligonucleotide that hybridizes specifically to a portion of the nucleic acid encoding a peripherin polypeptide) to an individual.
• routes of administration include, but are not limited to, oral, nasal, intravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal, intradermal, or topical administration.
• the amount of a composition administered will depend on many factors, including the route of administration and the formulation (e.g., solid, liquid, or emulsion).
• the method comprises administering to the individual an effective amount of an anti-peripherin antibody having specific binding affinity for a peripherin
• an imaging agent for example, P, Tc, In or I
• a suitable amount of an anti-peripherin antibody is any amount that is effective to image cells, for example, about 0.1 mCi to about 50.0 mCi.
• an effective amount of an anti-peripherin antibody may be an amount from about 0.01 mg to about 100 mg.
• Suitable methods of administering the imaging agent are as described above and can be targeted (e.g., to the brain) as described above. Methods of imaging are dependent upon the agent used and are well known to those of skill in this art.
• thermoregulatory sweat test and autoimmune serology were extracted and tabulated.
• Example 2 Rat Cell Lines
• mice nerve growth factor (2.5S; Alamone Laboratory, Cat No. N-100) was added on alternate days for 7 days (100 ng/mL).
• L6 skeletal muscle cells (ATCC, CRL-1458) were maintained in DMEM 4.5 supplemented with 10% horse serum.
• 2% fetal calf serum was substituted for 3 days; myotube formation was confirmed by microscopic examination.
• CG4 oligodendroglial-astrocytic progenitor cells (provided by Dr. Charles Howe, Department of Neurology, Mayo Clinic, Rochester, MN) were grown in proliferation medium and differentiated as described previously (Hinson et al, 2008, J. Exp. Med., 205:2473-81).
• Triton XI 00 permeabilized by exposing for 3 minutes to 0.05% Triton XI 00. All substrates were blocked with normal goat serum (10% in PBS; 15 minutes). Commercial antibody probes were diluted in block buffer. After applying primary antibodies (45 minutes), and washing, species-appropriate secondary antibodies were applied (45 minutes), substrates were rinsed, ProLong anti-fade medium (Molecular Probes P36935) was applied and coverslips were mounted. Individual sera yielding positive results were titrated in doubling dilutions to determine endpoints of antibody detection.
• Tissues from a female mouse were oriented in OCT and snap frozen in isopentane. Cryosections (8 ⁇ ) were air-dried, incubated sequentially in PBS containing 10% normal goat serum or 1% BSA (30 minutes), primary antibody
• PC12 cells (100 packed volume) were extracted in 2 mL buffer (0.15 M NaCl, 0.01 M NaP0 4 , 2 mM EDTA, pH 7.2) containing 1 % NP40, 0.1% SDS and protease inhibitors (CompleteTM, Roche 11697498001). After shaking (4°C, 1 hour), proteins were denatured and reduced by boiling (5 minutes) in 2% SDS and 2-mercaptoethanol, separated by electrophoresis in 10% polyacrylamide gel, and transferred to nitrocellulose for Western blot.
• Molecular weight standards included biotinylated broad range markers (BioRad 161-0322) or pre-stained SDS-PAGE standards (BioRad 161-0374).
• PC 12 lysate proteins were transb lotted to nitrocellulose. Bound antigenic protein was located by Western blot staining of excised vertical edge strips. The horizontal intervening strip bearing antigen, and a control horizontal strip lacking the antigen of interest, were probed with individual serum. Both were washed three times in high salt buffer (50 mM Tris-HCl, pH 7.4 containing 500 mM NaCl), and once in low salt buffer (100 mM NaCl). Bound IgG was eluted in glacial acetic acid, neutralized, dialyzed against PBS, 0.02%> sodium azide and applied to the composite mouse tissue substrate to evaluate the immunofluorescence staining pattern.
• high salt buffer 50 mM Tris-HCl, pH 7.4 containing 500 mM NaCl
• low salt buffer 100 mM NaCl
• sensorimotor neuropathy lumbosacral plexopathy, transverse myelitis, non-specified myelopathy, optic neuropathy, encephalitis, cerebellar ataxia and myasthenia gravis.
• Six individuals (23%) had one or more co-existing neural autoantibodies (voltage-gated neuronal calcium channel or potassium channel, GAD 65 [3 individuals], ganglionic acetylcholine receptor, CRMP-5).
• Dysautonomia was the most commonly documented clinical association (14 individuals; 54%). It was generalized in 2 and limited in 12; 8 had gastrointestinal (GI) dysmotility and 6 had abnormalities of sudomotor, cardiovagal or adrenergic functions. GI dysmotility was confirmed by endoscopy, manometry or transit studies.
• Endocrinopathy a marker autoantibody of endocrine autoimmunity or both.
• Endocrinologic disorders documented in 7 individuals, included diabetes (two individuals had autoantibodies specific for both GAD65 and islet antigen-2 [IA-2]), thyroid disorders, and premature menopause. Two individuals had thyroid autoantibodies (thyroperoxidase [2] or thyroglobulin [1]) without documented thyroid dysfunction.
• the peripherin-IgG increasingly is recognized as a biomarker for a subset of patients with inflammatory autoimmune CNS disorders that mimic multiple sclerosis (as epitomized by 3 of the original patients; numbers 11, 15 and 26 in Table 1).
• inflammatory autoimmune CNS disorders that mimic multiple sclerosis (as epitomized by 3 of the original patients; numbers 11, 15 and 26 in Table 1).
• MS mimic phenotype Nine patients with the "MS mimic phenotype" are described in Table 2.
• Table 1 Clinical-serological correlations in seropositive individuals
• CRMP collapsin response-mediator protein
• Peripherin-IgG a biomarker for a subset of inflammatory autoimmune CNS disorders presenting with predominant involvement of anterior visual pathways (optic nerve) and spinal cord (multiple sclerosis mimics), with and without endocrinopathy (all confirmed by Western blot)
• Figure 3 illustrates the distinctive pattern of human IgG immunostaining on mouse tissues.
• stomach upper panel
• immunoreactive elements were prominent in neural elements of smooth muscle (enteric ganglia and nerve trunks), mucosa and
• Immunoreactivity was prominent in nerve fibers surrounding islets of Langerhans in the pancreas, nerve fibers in interstitial tissue between thyroid follicles, and in nerve fibers adjacent to ovarian follicles. Immunoreactivity was not detected in liver.
• n 9;
• n 12.
• the peripherin-specific autoantibody can be a diagnostically useful marker of small fiber peripheral neuropathy / autonomic neuropathy in patients with type I diabetes and/or thyroid disease, and may be predictive for development of small fiber peripheral neuropathy in patients with endocrine

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